Mesenchymal stem cells (MSCs) are self-renewing multipotent cells capable of differentiating into several cell lineages including osteoblasts, chondrocytes, and adipocytes. Ryan, J. M. et al. Mesenchymal stem cells avoid allogenic rejection, J Inflamm (Lond), 2005. 2: p. 8. First described by Friedenstein et al. (Friedenstein, A. J., et al., Stromal cells responsible for transferring the microenvironment of the hemopoietic tissues. Cloning in vitro and retransplantation in vivo, Transplantation, 1974. 17(4): p. 331-40), MSCs have successfully been isolated from bone marrow (Friedenstein, A. J., et al.; Golde, D. W., et al., Origin of human bone marrow fibroblasts, Br J Haematol, 1980. 44(2): p. 183-7), adipose (Zuk, P. A., et al., Multilineage cells from human adipose tissue: implications for cell-based therapies, Tissue Eng, 2001. 7(2): p. 211-28; Iwashima, S., et al., Novel culture system of mesenchymal stromal cells from human subcutaneous adipose tissue, Stem Cells Dev, 2009. 18(4): p. 533-43), peripheral blood (Wagner, W., et al., Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood, Exp Hematol, 2005. 33(11): p. 1402-16), umbilical cord blood and matrix (Zeddou, M., et al., The umbilical cord matrix is a better source of mesenchymal stem cells (MSC) than the umbilical cord blood, Cell Biol Int.), fetal blood and liver (Campagnoli, C., et al., Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow, Blood, 2001. 98(8): p. 2396-402), connective tissue of dermis (Wagner, W., et al.; Lorenz, K., et al., Multilineage differentiation potential of human dermal skin-derived fibroblasts, Exp Dermatol, 2008 17(11): p. 925-32), and skeletal muscle sources (Wagner, W., et al.). The multi-differentiation potential of MSC raises a clinical interest to employ these cells for regeneration purposes, for example, in osteogenesis imperfecta. MSCs lack major histocompatibility complex class II antigens and have been shown in vitro to inhibit the activation and/or function of natural killer cells (Spaggiari, G. M., et al., Mesenchymal stem cells inhibit natural killer-cell proliferation, cytotoxicity, and cytokine production: role of indoleamine 2,3-dioxygenase and prostaglandin E2, Blood, 2008. 111(3): p. 1327-33), T cells (Meisel, R., et al., Human bone marrow stromal cells inhibit allogeneic T-cell responses by indoleamine 2,3-dioxygenase-mediated tryptophan degradation, Blood, 2004. 103(12): p. 4619-21; Aggarwal, S. and M. F. Pittenger, Human mesenchymal stem cells modulate allogeneic immune cell responses, Blood, 2005. 105(4): p. 1815-22; Bartholomew, A., et al., Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo, Exp Hematol, 2002. 30(1): p. 42-8), dendritic cells (Beyth, S., et al., Human mesenchymal stem cells alter antigen presenting cell maturation and induce T-cell unresponsiveness, Blood, 2005. 105(5): p. 2214-9; Jiang, X. X., et al., Human mesenchymal stem cells inhibit differentiation and function of monocyte-derived dendritic cells, Blood, 2005. 105(10): p. 4120-6; Nauta, A. J., et al., Mesenchymal stem cells inhibit generation and function of both CD34+-derived and monocyte-derived dendritic cells, J Immunol, 2006. 177(4): p. 2080-7), and B-cells (Corcione, A., et al., Human mesenchymal stem cells modulate B-cell functions, Blood, 2006. 107(1): p. 367-72). These immunomodulatory properties have led to clinical trials to assess their therapeutic potential for graft-versus-host disease after hematopoietic transplantation, type I diabetes, and multiple sclerosis. Due to easy access via liposuction, adipose has become the preferred source of MSCs for therapeutic applications.
Irrespective of their source, MSC isolation involves several steps including positive selection via the properties of plastic-adherence and colony formation (Uccelli, A., L. Moretta, and V. Pistoia, Mesenchymal stem cells in health and disease, Nat Rev Immunol, 2008. 8(9): p. 726-36). Although this eliminates contaminants such as blood and immune cells, a heterogeneous starting population and fibroblast contamination represent disadvantages. Fibroblasts are known to undergo senescence and apoptosis in culture, while surviving cells become immortal and potentially tumorogenic. Prockop, D. J. and S. D. Olson, Clinical trials with adult stem/progenitor cells for tissue repair: let's not overlook some essential precautions, Blood, 2007. 109(8): p. 3147-51. Thus, identification and elimination of fibroblasts from MSC culture could improve MSC yield and differentiation potential and also prevent tumor formation after MSC transplantation.
However, there are currently no markers which can be used to identify and isolate MSCs. Despite consensus that MSCs are positive for expression of CD73, CD90, and CD105, and negative for expression of hematopoietic cell surface markers CD11a, CD19, CD34, CD45, and HLA-DR (Horwitz, E. M., et al., Clarification of the nomenclature for MSC: The International Society for Cellular Therapy position statement, Cytotherapy, 2005. 7(5): p. 393-5), expression levels of these markers vary across laboratories due to tissue source or the specific culture conditions used. Uccelli, et al. Perhaps more importantly, fibroblasts also express CD105, CD73, and CD90 on their surface and lack hematopoietic markers. Covas, D. T., et al., Multipotent mesenchymal stromal cells obtained from diverse human tissues share functional properties and gene-expression profile with CD146+ perivascular cells and fibroblasts, Exp Hematol, 2008. 36(5): p. 642-54. Additionally, fibroblasts and MSCs share an almost identical in vitro morphology, rendering useless physical filtration techniques. Haniffa, M. A., et al., Mesenchymal stem cells: the fibroblasts' new clothes? Haematologica, 2009. 94(2): p. 258-63.